PROJECT SUMMARY In the oral environment, interactions between lectin-like adhesins on bacteria and complementary glycan motifs on mucosal epithelia and saliva-coated tooth surfaces play central roles in initial bacterial colonization. Lectin-glycan binding also is important for bacterial coadhesion, leading to the formation of multi-species microbial biofilms that have the potential to turn into pathogenic habitats causing dental, oral, and perhaps systemic disease. Considering (a) the multitude of bacterial species identified in oral biofilms, (b) the rich diversity of species-typical sialoglycans found on salivary glycoproteins, and (c) the innumerable glycan motifs expressed on the surface of other bacteria, it is likely that many more unidentified lectin-like bacterial adhesins exist in the oral microbiome, beyond the few ones currently known. In our preliminary studies, we have focused on a well characterized class of bacterial adhesins, the serine-rich protein (SRR) adhesins of oral streptococci. We were able to demonstrate that the glycan binding regions of these SRR proteins (SRR-BRs) can be forged by recombinant DNA techniques into highly specific probes for detecting and localizing glycan receptor structures in a variety of applications. We also identified hitherto unknown SRR-BRs that exhibit novel glycan-binding specificities. The overall objective of this project is to expand and greatly diversify the tool box of available SRR-BRs by harvesting novel SRR-BRs from the oral microbiomes of other mammals that express strikingly different sialoglycan termini. We will also use a diverse panel of human and animal red blood cells and synthetic glycan-functionalized particles to identify additional SRR-BRs with novel glycan-binding properties. All SRR-BRs created in this project will be tested and cross-validated for their usefulness as simple, affordable and easy-to-use and well-controlled tools to probe the glycomes of biological or pathological samples. This work will also greatly complement, expand and diversify prior efforts at tool generation initiated under earlier Common Fund mechanisms. The specific aims of the project are to 1. Harness previously identified SRR-BRs as glycan-binding probes and determine their glycan- binding specificities, using available glycan array technology. 2. Identify additional SRR-BRs with novel glycan-binding specificities to further expand the tool box of available glycan-binding molecules 3. Demonstrate the usefulness of novel SRR-BRs to probe glycomes of the oral environment, the blood circulation, and other diverse biological samples.